CAMP Measurement in Living Cells CNG Channels

One approach for real-time detection of cAMP changes is based on olfactory CNG cation channels (Rich et al. 2000). Increase in cAMP concentration leads to fast gating of the channels, and cAMP changes are monitored typically by imaging of the fluorescent Ca2+ indicator fura-2 or by electrophysiological recordings of Ca2+ currents. The use of wild-type CNG channels to detect changes in cAMP concentration shows several limitations. First, wild-type channels have a lower affinity for cAMP than for cGMP (Dhallan et al. 1990; Rich et al. 2001b). Second, CNG channels can also be opened directly by nitric oxide (Broillet 2000). Third, the opening of these channels is strongly inhibited upon Ca2+-calmodulin binding (Liu et al. 1994). To surmount some of these limitations, the properties of the wild-type CNG channels have been modified (Rich et al. 2001a). To reduce the sensitivity to cGMP and increase the sensitivity to cAMP, two mutations, C460W and E583M, have been introduced, whereas the regulation of the channel by Ca2+-calmodulin has been removed by deletion of residues 61-90. Thus, the channel A61-90/C460W/E583M is virtually insensitive to cGMP and sensitive to low cAMP concentrations (Rich et al. 2001a). Although these improvements have extended the application of the CNG-based probes, some limitations persist. Firstly, changes in cAMP concentration can be detected only at the plasma membrane, where the probe is located, and the cAMP signal cannot be revealed in other compartments. Secondly, overexpression of the channel and the consequent increase in Ca2+ influx may affect the intra-cellular concentration of cAMP by acting on Ca2+-sensitive ACs or PDEs. As a consequence, the concentration of cAMP reported by the CNG-based sensor may be non-physiological.

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